| Purpose: To observe the effect of electroacupuncture(EA)at “Zusanli”(ST 36)on energy metabolism and mitochondrial dynamics related indicators of skeletal muscles in spleen-qi deficiency model rats,so as to clarify the mechanism of EA regulating mitochondrial dynamics in the treatment of spleen-qi deficiency syndrome,and partially revealing the biological basis of ST 36 in strengthening spleen and replenishing qi.Exploring the functional essence of traditional Chinese medicine theory “the spleen governs the muscles” from a new perspective of mitochondrial dynamics,which may partially reveal the scientific connotation of “the spleen governs the muscles”.Material and method: 1.Experimental animals: Thirty-two male SD rats,SPF grade,weighing 200 ± 10 g,were allowed to eat and drink freely.After 1 week of adaptive feeding,the experiment was started.The rats were randomly divided into 4 groups: normal control group(the normal group);the spleen-qi deficiency model group(the model group);the EA at ST 36 group(the ST 36 group);the EA at non-acupoint group(the non-acupoint group),n=8 rats in each group.2.Modeling method and evaluation standard The spleen-qi deficiency model was established by improper diet and exhaustive swimming.Briefly,rats underwent 48 h of food deprivation,24 h of unlimited intake,and loaded swimming.The rats swam with a load of steel rings attached to their tails;the combined weight of the rings was approximately 5% of each rat's body weight.The rats were individually placed in a columnar swimming pool(height of 65 cm,radius of 20 cm)for 20 min,using 40 cm of water height;the water was maintained at 37oC.Each rat was subjected to loaded swimming every day.The improper diet and exhaustive swimming regimen were performed for five total cycles(3 days per cycle).At the end of five cycles,the spleen-qi deficiency rats exhibited low appetite,fatigue,dull or lusterless hair,and reduced body weight.These symptoms met the diagnosis criteria for spleen-qi deficiency,as defined inMethodology of Establishing Experimental Animal Models for Traditional Chinese Medicine Research,which suggested that the animal model was established successfully.3.Intervention methods Following successful model establishment,the ST 36 group received EA at bilateral ST 36;the stainless-steel needles(0.19 × 10 mm)were perpendicularly inserted to a depth of 5 mm and connected with the acupuncture stimulator SDZ-II.The disperse-dense wave,with an intensity of 0.5 m A and frequency of 2 Hz/15 Hz was used,and the stimulation lasted for 20 min.The non-acupoint group received EA at bilateral non-acupoints(20 mm lateral to the midline on the back and 15 mm above the iliac crest connecting line);the needle was inserted horizontally to a depth of 5–8 mm with the same electrical stimulation as described in ST36 group.Treatments were repeated daily for 7 days.Rats in the control and model groups were restrained in the same manner,but did not receive any treatment.4.Detection Indicators(1)General observation: The general state of the rats was observed daily,including the state of the rats' mental state,activity level,fur color,and fecal morphology.(2)Body weight measurement: The rats' body weight was measured every 3 days from the day before the model was established.The final body weight was measured after the last treatment,and the changes were recorded and counted.(3)Measurement of food intake: The average food intake of each group of rats was weighed and calculated on days 4,7,10,13,16,19,and 22 of the experiment.(4)Determination of the grasping force: The forelimb grasping force was measured using a YLS-13 A mouse grip tester;(5)Determination of ATP content: The colorimetric method was used to detect the ATP content in rats skeletal muscle tissue.(6)Determination of mitochondrial respiratory chain complex II and IV activity: The activity of mitochondrial respiratory chain complex II and IV in skeletal muscle tissue of rats was detected by enzyme-labeled method.(7)The ultrastructure changes of skeletal muscle tissue of rats were observed by transmission electron microscopy,and the mitochondrial fusion and mitotic conditions were observed.(8)The expression of Mfn1,Mfn2,Opa1,Drp1,Fis1 m RNA in rats skeletal muscle tissue was measured by fluorescence quantitative PCR.(9)Western blot was used to determine the expression of Mfn1,Mfn2,Opa1,Drp1 and Fis1 proteins in skeletal muscle tissue of rats.Results: 1.General observation of rats in each group The rats in the normal group were physically strong,with good mental state,shiny fur,high excitability and normal stool.Rats in the model group began to lose weight gradually from the third day of model building,reduced food intake,poor spirit,dry coats and fall off,lazy movement and loose stools.After EA at ST 36,the activity of the rats,the food intake,the body weight,the mental state,the fur dropping and the stools were all improved.The general state of the rats in the non-acupoint group did not improve significantly compared with the model group.2.Body weight of rats in each group Before modeling,the body weight of all groups was basically the same,with no significant difference(P>0.05).From the second modeling cycle,body weight of rats in the model group,ST 36 group and non-acupoint group were significantly lower than those in the normal group(P<0.05).After modeling,the weight of rats in the three groups increased gradually.After treatment,the weight of rats in the ST 36 group was significantly higher than that of the model and the non-acupoint groups(P<0.05),while the weight of rats in the non-acupoint was not significantly different from that in the model group(P > 0.05).3.Food intake of rats in each group After the second modeling cycle,the food intake of rats in the model,the ST 36 and the non-acupoint groups increased slightly compared with the previous modeling period,but from the third cycle,the food intake gradually decreased.With the prolongation of modelling time,the declining trend of food intake in the three groups has become increasingly apparent.After modeling,the food intake of rats in the three groups of increased gradually,but it was still less than that in the normal group.After the treatment,the intake of rats in the ST 36 group was increased compared with the model group and the non-acupoint group.4.Grip strength of rats in each group There was no significant difference in the grip strength of the rats before the modeling(P>0.05).After the modeling,the grip strength of the model group,the ST 36 group and the non-acupoint group were lower than that of the normal group(P<0.05).Compared with the model group,the grip strength of the ST 36 group was significantly increased(P<0.05),while the non-acupoint group had no significant change in the grip strength(P>0.05).The differences between the non-acupoint group and the ST 36 group were significant(P<0.05).5.ATP contents in skeletal muscle tissue of rats in each group Compared with the normal group,the ATP contents in skeletal muscle tissues of rats in the model group and the non-acupoint group were decreased significantly(P < 0.05).Compared with the model and non-acupoint groups,the ATP contents in the skeletal muscle tissue of rats in the ST 36 group were significantly increased(both P < 0.05),but there were no statistically significant differences between the non-acupoint group and the model group(P > 0.05).6.Mitochondrial respiratory chain enzyme II and IV activities in skeletal muscle tissue of rats in each group Comparison of mitochondrial respiratory function in rats showed that complex II and IV activities in the model group were significantly lower than those in the normal group(P < 0.05).Further,EA at ST36 can restore the decrease in complex II and IV activities to normal levels(P < 0.05).However,respiratory complex II and IV activities were not significantly different between the model and non-acupoint groups(P > 0.05).7.Skeletal muscle ultrastructure and mitochondrial fission and fusion in rats of each group Transmission electron microscopy was used to observe the ultrastructure changes of rats skeletal muscle tissue and mitochondrial fission and fusion in four groups.Results: In the normal group,skeletal muscle fibers were arranged in an orderly fashion;mitochondria were observed undergoing both fission and fusion.In contrast,rats in the model group exhibited a large number of muscle fibers that were ruptured,damaged,and disorganized;moreover,many vacuoles with different sizes,and abnormally shaped or swollen mitochondria,were observed in the model group.ST 36 treatment improved the disordered fiber arrangement,and reduced the population of damaged mitochondria;thus,fused and elongated mitochondriawere readily observed.Compared with the model group,there were no obvious improvements in the non-acupoint group.8.Comparison of the expression of Mfn1,Mfn2,Opa1,Drp1 and Fis1 m RNA in skeletal muscle tissue of rats in each group The m RNA expression of Mfn1,Mfn2,Opa1 and Drp1 was significantly lower in the model group,compared with the normal group(all P < 0.05).Moreover,m RNA expression of Mfn1,Mfn2,Opa1 and Drp1 was significantly higher in the ST36 group(all P < 0.05);however,there were no significant changes in the non-acupoint group,compared with the model group(all P > 0.05).The expression of Fis1 m RNA in the model group,ST 36 group and non-acupoint group decreased compared with the normal group,but the difference was not statistically significant(P > 0.05).9.Comparison of the expression of Mfn1,Mfn2,Opa1,Drp1 and Fis1 protein in skeletal muscle tissue of rats in each group Compared with the normal group,the relative expressions of Mfn1,Mfn2,Opa1 and Drp1 were significantly decreased in the model(all P < 0.05).The relative expressions of the above proteins in the ST 36 group were higher than in the model group(all P < 0.05);however,the non-acupoint group did not show significant differences.There was no significant difference in the expression of mitochondrial fission protein Fis1 in the model group,the ST 36 group and the non-acupoint group compared with the normal group(P > 0.05).Conclusion: 1.Acupuncture at ST 36 can improve the mitochondrial respiratory chain complex II and IV activities,increases ATP contents,and increase mitochondrial energy metabolism level,so as to play the role of "strengthening spleen and replenishing qi",thereby improving the body thinning,eating less,fatigue and other symptoms caused by spleen-qi deficiency;2.Acupuncture at ST 36 can improve the imbalance of mitochondrial fission and fusion of skeletal muscles in rats with spleen-qi deficiency,thereby improving the structure and function of mitochondria and increasing the level of energy metabolism;3.Non-acupoints can not effectively improve the above indicators,further confirming themeridian-acupoints specificity of the ST 36. |
PDF Full Text Request